Studies of plasma membrane mechanics and plasma membrane-cytoskeleton interactions using optical tweezers and fluorescence imaging.

We use optical tweezers in conjunction with an optical position-sensing system, which spectrally filters signals generated by a trapped fluorescent microsphere to study plasma membrane (PM) mechanics and its interactions with cytoskeleton. We dynamically measure the PM tethering force on human embryonic kidney cells that are a standard cultured cell line. Recorded tethering force vs. PM displacement profiles, revealed the tether formation process, initiated with linear deformation of the PM, followed by a nonlinear regime and terminated with the local separation of PM. Tethering force vs. displacement profiles were used to estimate tether formation force and stiffness parameter of the PM. Integration of the force-displacement profiles yielded the work of tether formation, including linear and nonlinear components. Our results demonstrate that spectral filtering of the optically trapped fluorescent microsphere image formed on the position-sensing system overcomes the artifacts introduced by the transillumination imaging and allows accurate measures of PM mechanics before and during the initial stages of tether formation.

[1]  D. Frenkel,et al.  Force barriers for membrane tube formation. , 2005, Physical review letters.

[2]  Evan Evans,et al.  Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. I. Membrane separation from the cytoskeleton. , 2005, Biophysical journal.

[3]  J. Mege,et al.  Use of cell contour analysis to evaluate the affinity between macrophages and glutaraldehyde-treated erythrocytes. , 1987, Biophysical journal.

[4]  E. Evans,et al.  Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. II. Tether flow terminated by P-selectin dissociation from PSGL-1. , 2005, Biophysical journal.

[5]  Bahman Anvari,et al.  Combining optical tweezers and patch clamp for studies of cell membrane electromechanics. , 2004, The Review of scientific instruments.

[6]  J. Hoh,et al.  Surface morphology and mechanical properties of MDCK monolayers by atomic force microscopy , 1996 .

[7]  Gary J. Brouhard,et al.  Advanced optical tweezers for the study of cellular and molecular biomechanics , 2003, IEEE Transactions on Biomedical Engineering.

[8]  K. Nagayama,et al.  Local mechanical properties measured by atomic force microscopy for cultured bovine endothelial cells exposed to shear stress. , 2000, Journal of biomechanics.

[9]  W. Brownell,et al.  Effects of salicylate on plasma membrane mechanics. , 2005, Journal of neurophysiology.

[10]  A. Mehta,et al.  Reflections of a lucid dreamer: optical trap design considerations. , 1998, Methods in cell biology.

[11]  W. Brownell,et al.  Micropipette aspiration on the outer hair cell lateral wall. , 1997, Biophysical journal.

[12]  Feng Qian,et al.  Optical tweezers study of viscoelastic properties in the outer hair cell plasma membrane , 2004, SPIE BiOS.

[13]  E. Sackmann,et al.  A micromechanic study of cell polarity and plasma membrane cell body coupling in Dictyostelium. , 2000, Biophysical journal.

[14]  S. Chu,et al.  Quantitative measurements of force and displacement using an optical trap. , 1996, Biophysical journal.

[15]  R. Hochmuth,et al.  Membrane tethers formed from blood cells with available area and determination of their adhesion energy. , 2002, Biophysical journal.

[16]  W. C. Hwang,et al.  Energy of dissociation of lipid bilayer from the membrane skeleton of red blood cells. , 1997, Biophysical journal.

[17]  M W Berns,et al.  Parametric study of the forces on microspheres held by optical tweezers. , 1994, Applied optics.

[18]  R. Hochmuth,et al.  Micropipette aspiration of living cells. , 2000, Journal of biomechanics.

[19]  Robert C. Gauthier,et al.  Optical levitation of spheres: analytical development and numerical computations of the force equations , 1995 .

[20]  Bahman Anvari,et al.  Dynamic Measurements of Transverse Optical Trapping Force in Biological Applications , 2004, Annals of Biomedical Engineering.

[21]  R M Nerem,et al.  Micropipette Aspiration of Cultured Bovine Aortic Endothelial Cells Exposed to Shear Stress , 1987, Arteriosclerosis.

[22]  W. Brownell,et al.  Membrane tether formation from outer hair cells with optical tweezers. , 2002, Biophysical journal.

[23]  S. Chu,et al.  Observation of a single-beam gradient force optical trap for dielectric particles. , 1986, Optics letters.